The purpose of this proposal is to study molecular mechanisms of adenosine-regulated glutamate signaling, which is an essential component of the medial prefrontal cortex (mPFC)-striatal circuit and ethanol seeking behaviors. We will utilize a unique and highly integrated experimental approach, which includes novel transgenic mice and optogenetics, which will contribute to elucidating the role of adenosine signaling in alcohol use disorders. During the previous funding period, our studies revealed that ENT1 (equilibrative nucleoside transporter type 1; Slc29a1), an ethanol-sensitive adenosine transporter, regulates adenosine and glutamate signaling in the striatum and plays a pivotal role in neuron-glial interaction to regulate ethanol-seeking behaviors. Our recent findings demonstrated that genetic and pharmacological inhibition of ENT1 in the dorsomedial striatum (DMS) promote goal- directed behaviors through hypo-adenosine A2A receptor function, which contributes to increased ethanol seeking behaviors in an operant conditioning experiment. However, it is unclear whether astrocytic or neuronal ENT1 regulates adenosine levels and adenosine receptor-mediated signaling in the mPFC-DMS circuits. Our main hypothesis is that astrocytic ENT1-regulated adenosine signaling plays an essential role in glutamate neurotransmission in the cortico-striatal circuits and ethanol consumption. To investigate this hypothesis, we propose three aims. First, we will examine whether inducible expression of ENT1 regulates adenosine and glutamate signaling in the cortico-striatal circuits. Using recently generated tetracyline-dependent inducible ENT1 transgenic mice, we will examine whether overexpression of ENT1 alters adenosine and glutamate signaling in the striatum and prefrontal cortex as well as ethanol-seeking behaviors. In this aim, we will utilize CAG (universal promoter) driven rtTA (reverse tetracycline controlled transactivator) expressing mice to examine overexpression of ENT1 in the brain. We will investigate whether ENT1-dependent signaling in the two distinct striatal regions (NAc and DMS) and mPFC alters ethanol-seeking behaviors. Second, using astrocyte or neuron-specific ENT1 overexpressing mice, we will investigate whether astrocyte or neuronal ENT1 regulates ethanol-seeking behaviors. Using astrocyte (Gfap) or neuron-specific (CamK2a) rtTA expressing mice, we plan to investigate whether astrocyte- or neuron-specific ENT1 regulates goal-directed and ethanol seeking behaviors. In addition, we will examine whether agonists or antagonists of adenosine receptors regulate ethanol-seeking behaviors. Third, we will investigate ethanol-seeking behaviors using circuit-specific optogenetics. Our preliminary study suggests that inhibition of the A2AR-containing striatopallidal circuit in the DMS promotes ethanol-seeking behavior in an operant chamber. To examine whether optogenetic regulation of striatopallidal neurons in the DMS regulates ethanol-seeking behaviors, we will use laser-stimulated channelrhodopsin (ChR2) and halorhodopsin (eNpHR3) under the control of the A2AR promoter. These studies will elucidate molecular mechanisms underlying adenosine-regulated glutamate signaling in both neuron-glial interactions and cortico-striatal circuits and their role in ethanol-seeking behaviors.